Cation-exchange purification of mutagenized bovine beta-casein expressed in transgenic mouse milk: Its putative Asn(68)-linked glycan is heterogeneous

Citation
Bk. Choi et al., Cation-exchange purification of mutagenized bovine beta-casein expressed in transgenic mouse milk: Its putative Asn(68)-linked glycan is heterogeneous, J DAIRY SCI, 84(1), 2001, pp. 44-49
Citations number
26
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Food Science/Nutrition
Journal title
JOURNAL OF DAIRY SCIENCE
ISSN journal
0022-0302 → ACNP
Volume
84
Issue
1
Year of publication
2001
Pages
44 - 49
Database
ISI
SICI code
0022-0302(200101)84:1<44:CPOMBB>2.0.ZU;2-V
Abstract
Bovine beta -casein (A(2) genetic variant) was mutagenized to (L70S/P71S) a nd expressed in transgenic mouse milk. This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X -S/T) (3). Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it. This novel protein was purified from tra nsgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatogr aphy (FPLC). The novel beta -casein was separated without cross contaminati on from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2 beta -mercaptoethanol. The purified (L 70S/P71S) beta -casein showed an N-linked oligosaccharide attached to Asn(6 8) and different lectin binding profiles compared with the same protein exp ressed in yeast. The mouse-expressed beta -casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated beta -casein e xpressed in Pichia pastoris (4). In addition, the five serine residues loca ted at amino terminus of wild type bovine beta -casein were shown to be nor mally phosphorylated as in native bovine beta -casein.